Luteolin and diosmin/diosmetin as novel stat3 inhibitors for treating autism

ABSTRACT

The present invention includes methods for the treatment of autoimmune disorders such as autism, schizophrenia, and type 1 diabetes. Flavonoids, luteolin, diosmin, and diosmin&#39;s aglycone form, diosmetin, were found to inhibit activation/phosphorylation of STAT3 induced by IL-6 in cultured neuronal cells. Furthermore, mice treated with diosmin showed a significant reduction of autistic phenotype induced by IL-6 through inhibition of STAT3 activation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior filed InternationalApplication, Serial Number PCT/US2009/062753, entitled, “Luteolin andDiosmin/Diosmetin as Novel STAT3 Inhibitors for Treating Autism” filedOct. 30, 2009, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/109,538 entitled, “Luteolin andDiosmin/Diosmetin as Novel STAT3 (Signal Transducer and Activator ofTranscription) Inhibitors for Treating Autism”, filed Oct. 30, 2008,which are herein incorporated by reference.

FIELD OF INVENTION

The present invention relates to compositions and methods for thescreening and treatment of autoimmune disorders such as autism,schizophrenia, and type 1 diabetes. Specifically this invention usesflavonoids to inhibit the activation/phosphorylation of STAT3 induced byIL-6.

BACKGROUND OF THE INVENTION

Autism is a brain disorder that begins in early childhood and persiststhroughout adulthood. It affects three crucial areas of development:communication, social interaction, and creative or imaginative play.Children with autism have difficulties in social interaction andcommunication and may show repetitive behaviors and have unusualattachments to objects or routines. Similar to other neuropsychiatricdisorders, the mechanism(s) involved in autism development have yet tobe fully determined. Studies have indicated brain development in autismis abnormally characterized by accelerated growth in early life thatresults in brain enlargement in childhood (Aylward et al., 2002;Courchesne et al., 2001; Tate et al. 2007). Accelerated brain growth isalso a feature of acquired macrocephaly, a finding in many autisticchildren. Also along these lines, a positive association betweenincreasing radiate white matter volume and motor skill impairment inchildren with autism has also been shown (Mostofsky et al. 2007).Although such results are not universal for all autistic patients, theydo define a significant subgroup of affected patients (Acosta and Pearl2004; Ben Bashat et al. 2007), which potentially could be spared diseaseif intervention could be made early in neurodevelopment.

Autism affects thousands of Americans and encompasses a number ofsubtypes, with various putative causes and few documented ameliorativetreatments. The disorders of the autistic spectrum may be present atbirth, or may have later onset, for example, at ages two or three.Autism has increased tenfold in the last fifteen years. It is estimatedto afflict between 2 and 5 of every 1000 children and is four times morelikely to strike boys than girls. It is estimated that about 1.5 millionAmerican children and adults have autism. The U.S. Department ofEducation has estimated the rate of autism is increasing at 10 to 17percent each year.

Autism has a strong genetic component, and in some families, autismtends to be more prevalent. In identical twins with autism both areusually affected. However, the number of children with autism appears tobe increasing more than expected for a genetic disorder. This suggeststo scientists that genetic abnormalities require the influence of otherfactors to cause the disorder.

The main goals of treatment are to lessen associated deficits and familydistress, and to increase quality of life and functional independence.No single treatment is best and treatment is typically tailored to thechild's needs. Although there is no cure for autism, clinicians do agreethat early detection could significantly improve chances in if notpreventing it, then at last reducing its weakening effects. Currenttreatments were shown efficacious when applied earlier in child's life,prior to the full blown onset. Thus the early prognosis is a vitalcomponent in the fight against this disease.

IL-6 and JAK2/STAT3

Inflammation plays a key role in many neurological diseases as well asin neurodegenerative diseases displaying cognitive and behavioralimpairments. Recent studies have shown that abnormal immune responsescontribute to autistic pathogenesis in animals (Warren et al., 2005).Neuroglia inflammation has been observed in the brain tissue of autisticindividuals from as young as 5 to as old as 45 (Honda, Michelle (2008)Flavonoids benefit oxidative stress and inflammation associated withautism, www.MichelleHonda.com) Studies have been conducted that showthat autistic brains show evidence of inflammation in certain brainareas, most prominently in the cerebellum. Ongoing inflammation was alsofound in the fluid surrounding the brain and spinal cord (Vargas D. L.,et al. (2004) Neuroglial activation and neuroinflammation in the brainof patients with autism, Annals of Neurology 57:67-81).

Sharp increases in IL-6 production have been shown under inflammatoryconditions in the brain. The main sources of IL-6 are reactiveastrocytes and microglia (Behrens, M. M. et al. (2008) Interleukin-6mediates the increase in NADPH-oxidase in ketamine model ofschizophrenia, The Journal of Neuroscience, 28(51):13957-13966).Interleukin 6 (IL-6) plays a key role in developing autistic phenotypein the mice (Smith et al., 2007). It is well known that IL-6 highlyactivates signal transducer and activator of transcription protein 3(STAT3) signal pathway in vitro and in vivo (Ebong et al., 2004; Ogataet al., 1997). The binding of IL-6 to its receptor (IL-6R) generates acomplex with the gp130 protein thus triggering activation ofJanus-activated kinases (JAK). This is then critical for inducingphosphorylation of docking sites of the receptor for SHP2 (SH2domain-containing tyrosine phosphatase) and STAT (signal transducers andactivators of transcription) proteins (Heinrich et al., 1998). There arevarious STAT protein variants, which cause an array of signaling whichcan result in neuronal death (Giunta et al., 2006, 2007, & 2008) orgrowth/differentiation (Kamimura et al., 2003). Phosphorylated STAT3molecules dimerize and then move to the nucleus, where they lead totransactivation of target genes largely associated with neuronal growth(Schindler & Darnell 1995).

JAK2/STAT3 pathways induced by IL-6 play a crucial role in cellproliferation, a phenomenon known to occur at excess levels in thebrains of a significant proportion of autistic children. Various studiesdemonstrate IL-6 signaling through Stat3 modulates the growth andproliferation of both neurons directly or via inhibition of neurondeath. In vitro, stimulated astrocytes and microglia produce IL-6 (VanWagoner et al., 1999; John et al., 2003), and its chimeric derivativesrescue neurons preserve myelin basic protein production in hippocampalslices exposed to excitotoxic insult (Pizzi et al., 2004). IL-6 alsosupports the survival of cultured postnatal rat septal cholinergicneurons (Hamma et al., 1999) Moreover, IL-6 has been shown to becrucially involved in the development, differentiation, regeneration,and degeneration of neurons in the peripheral and central nervous system(Gadient & Otten, 1994 & 1997). Further, evidence to support IL-6/Stat3mediated growth signaling was demonstrated in spinal cord-derived neuralprogenitor cells (NPCs) exposed to IL-6 and epidermal growth factor(EGF). The phosphorylation level of Jak2/Stat3 was determined andmaximal phosphorylation occurred at 30 minutes of NPCs exposure to IL-6.In addition, phosphorylation of Jak2/Stat3 was attenuated bypre-treatment of cells with AG490, the JAK2 specific inhibitor,suggesting that this pathway play a key in repopulation and regenerationof spinal cord tissue after injury (Kang et al., 2008).

Maternal Infection Activation

The earliest point in neurodevelopment is fetal. During theneurodevelopment stage, it has been reported that activation/regulationof STAT3 signal pathways is critically involved (Yvonne et al. 2000).Maternal infection increases the risk of fetal neurological injury.Systemic inflammatory response during pregnancy may contribute toneuropsychiatric disease in childhood and adulthood (Huleihel et al.,2004). Epidemiological studies have shown that the risk of developingautism is associated with prenatal maternal infection. This presumablyresults from neurodevelopmental defects triggered by cytokine-relatedinflammatory events (Ashdown et al., 2006; Meyer et al., 2008), as thereis increased incidence of autism in the offspring of mothers whosuffered infections while pregnant (Patterson, 2005). Because of thestrong genetic component in autism, it is likely that only geneticallysusceptible individuals who were exposed to maternal infection woulddevelop the disorder. Thus, it has been suggested that the risk ofautism associated with maternal infection may be considerably more thanthree to seven-fold in susceptible individuals.

Several studies suggest the maternal immune response or “activation” toinfection, rather than infection of the fetus itself (Shi et al., 2003 &2005), is responsible for the increased incidence of autism in theoffspring of mothers who suffered infections while pregnant. Aftermaternal immune activation (MIA) by influenza, LPS, or poly(I:C),cytokine levels are altered in the maternal serum as well as theamniotic fluid, placenta, and, most importantly, the fetal brain (Fidelet al., 1994; Cai et al., 2000; Urakubo et al., 2001; Gayle et al.,2004; Paintlia et al., 2004; Gilmore et al., 2005; Beloosesky et al.,2006; Meyer et al., 2006). Cytokines were initially characterized ascompounds of the immune response, but have since been found to play amuch broader, diverse role in physiology. Cytokines, includinginterleukin-6 (IL-6), IL-11, IL-27 and leukemia inhibitory factor, havebeen shown to confer signaling in both the developing and adult brain;both directly and indirectly modulating neuronal functions (Bauer etal., 2007). Therefore, it is not surprising that inflammatory cytokinesand their receptors modulate brain morphology during development aswell. Particularly, IL-6 is likely a central link between maternalinfection and neurodevelopmental derangement (Akira et al., 1990; Smithet al., 2007).

Maternal MIA in pregnant rodents yields offspring with abnormalities inbehavior, histology, and gene expression that mimic psychiatric featuresof autism. This suggests MIA as a useful model of the development ofautism. Specifically, single maternal injection of IL-6 on day 12.5 ofpregnancy caused prepulse inhibition (PPI) and latent inhibition (LI)deficits in adult offspring. Co-administration of anti-IL-6 in the poly(I:C) model of MIA prevented the PPI, LI, and exploratory and socialdeficits caused by poly(I:C) and normalized the associated changes ingene expression in the brains of the adult offspring. Most convincingly,MIA in IL-6 knock-out mice did not yield several of the behavioralchanges observed in the offspring of wild-type mice after MIA.

MIA-induced cytokines confer both direct and indirect effects on thefetus. Two methods have been used to study these effects: injecting orup-regulating cytokines during pregnancy in the absence of MIA, orblocking endogenous cytokines or preventing their induction during MIA.A study of the role of TNF-α in LPS-induced fetal loss and growthrestriction indicated injection of anti-TNF-α antibodies or an inhibitorof TNF-α synthesis [pentoxifylline] can reduce these effects of LPS.Conversely, injection of TNF-α alone can induce fetal loss (Siler etal., 1994 and Xu et al., 2006) which is significantly worse in IL-18knockout mice, but not in IL-1α/β knockout mice (Wang et al., 2006).

Much of the previous investigations of cytokine mediation of MIA effectson neuropathology and behavior in the offspring have focused on IL-6.This cytokine is involved in the regulation of physiological processesincluding inflammation and neurodevelopment; making it a particularlyappealing candidate molecule for MIA-induced neuropathology. Indeed,during neurodevelopment, the signal transducer and activator oftranscription-3 (STAT3) pathway, activated by IL-6, maintainshomeostasis between neuro- and gliogenesis (He et al., 2005 and Murphyet al., 2000).

In support, Samuelsson et al., 2006 intraperitoneally injected IL-6 inpregnant rats for 3 days resulting in severe effects on the offspring.An important finding was that IL-6 mRNA levels remain elevated in thehippocampi of the offspring at 4 and 24 weeks of age; indicative of theongoing state of immune dysregulation in adult autistic brains. Spatialmemory in the water maze, a hippocampal-dependent behavior, was observedin that study. Importantly, the IL-6-treated offspring displayedincreased latency to escape and time spent near the pool wall.Therefore, prolonged exposure to elevated IL-6 in utero causes a deficitin working memory (for reviews see Patterson 2008).

Blocking endogenous IL-6 in MIA also supports the central role of thiscytokine (Smith et al., 2007). Co-injection of anti-IL-6 antibody withmaternal poly(I:C) blocks the effects of MIA on the behavior of theoffspring. Further, maternal injection of poly(I:C) in an IL-6 knockoutmouse results in normal behaving offspring. In addition, the anti-IL-6antibody also blocks the changes in brain transcription induced bymaternal poly(I:C) (Patterson 2008). Maternal injection of poly(I:C)induces expression of IL-6 mRNA in fetal brain and placenta, and this isalso dependent on the IL-6 induced by maternal poly(I:C) (Patterson2008) (E. Hsiao and P. H. Patterson, unpublished). Taken together theseprevious works by other groups indicate both direct and indirect(positive feedback loop) mechanisms for IL-6 mediated MIA in the contextof aberrant fetal brain development which could lead to an autism-likephenotype (Patterson 2008).

Schizophrenia

IL-6 plays a role not only in the pathogenesis of autism but also in thepathogenesis of schizophrenia in the context of maternal immuneactivation. MIA is used to describe an increase in circulating maternalcytokines in response to an infection during pregnancy (Smith et al.,2007). The ensuing cytokine response and the highly susceptibledevelopmental period in which it occurs may precipitate theneuropathological and behavioral deficits observed in autism and relateddisorders such as schizophrenia. Schizophrenia and autism can resultfrom the interaction between a susceptibility genotype and environmentalrisk factors. IL-6 is critical for mediating the behavioral andtranscriptional changes in offspring in which mothers were exposed toinfection. There is evidence to indicate that the maternal immuneresponse, as opposed to the direct infection of the fetus, isresponsible for the increased incidence of schizophrenia and autism inthe offspring of mothers who suffer infections during pregnancy (Smithet al., 2007).

Neuroprotection IL-6/STAT3 signaling against N-methyl-D-aspartate(NMDA)-induced neurotoxicity has also been demonstrated. Culturedcerebellar granule neurons (CGNs) from postnatal (eight-day) infant ratswere chronically exposed to IL-6 for eight days, and then NMDA (100mol/L) was administered to the cultured CGNs. By way of MTT, and TUNELassays, as well as confocal laser scanning microscope (CLSM) and westernblotting, it was shown that NMDA stimulation of cell death could beavoided by IL-6 treatment. The NMDA stimulation of the CGNs chronicallypretreated with IL-6 caused a very large increase in neuronal vitality,as well suppression of neuronal apoptosis compared with that in thecontrol neurons without IL-6 pretreatment. The levels of phospho-Stat3are significantly higher in IL-6-pretreated CGNs than those inIL-6-untreated neurons. Ketamine is an NMDA receptor antagonist whichproduces psychosis in humans and exacerbates symptoms in schizophrenicpatients. Ketamine has been shown to activate the innate immune enzymeNADPH-oxidase in the brain. IL-6 has been shown to be the downstreammediator of ketamine in the induction of Nox2 and to activate NADPHoxidase in the brain providing further evidence for the role of IL-6 inthe pathogenesis of schizophrenia (Behrens et al. 2008).

Diabetes

There is also an interaction between IL-6 and insulin systems,specifically insulin-like growth factor-1 (IGF-1) (Venkatasubramanian,G. (2007) Pathogenesis of Schizophrenia & Autism: The Interactionbetween Interleukin and Insulin Systems, Journal of Neuroscience 27(40):10695-10702). IGF-1 has a significant role in fetal development and hasneuroprotective, anti-apoptotic properties that are crucial for theoptimal development of the brain (Fowden, 2003; Dore et al., 1997). IL-6inhibits the secretion and biological activity of IGF-1 (de Martino etal., 2000; Lazarus et al., 1993). Cerebral damage in fetalpro-inflammatory states is associated with high IL-6 and low IGF-1levels (Hansen-Pupp et al., 2007). Elevated IL-6 and decreased IGF-1levels are also shown in schizophrenia and autism (Potvin et al., 2007;Venkatasubramanian et al., 2007; Jyonouchi et al., 2001; Riikonen etal., 2006).

Patients with diabetes also have reduced serum levels of IGF-1 thatoccur in response to a state of insulin resistance. IGF-1 acts inconcert with insulin and has an important role in maintaining glucosehomeostasis and protein metabolism in type 1 diabetes (Simpson, H. L. etal. (2004) Insulin-like growth factor has a direct effect on glucose andprotein metabolism, but no effect on lipid metabolism in type 1diabetes, The Journal of Clinical Endocrinology & Metabolism89(1):425-432). In addition, similarly to the levels shown inschizophrenia and autism, patients exhibiting insulin-resistant statessuch as in diabetes have been shown to have increased levels of IL-6 anddecreased levels of IGF-1 (Glund, S. et al. (2007) Inerleukin-6 directlyincreases glucose metabolism in resting human skeletal muscle, TheJournal of the American Diabetes Association 56(6):1630-1637).

Flavonoids

Flavonoids, plant polyphenolic compounds abundant in fruits andvegetables, exhibit a wide variety of biological effects, includingantioxidant free-radical scavenging and anti-inflammatory properties(Rice-Evans C, Packer L (1998) in Flavonoids in Health and Diseases, edsRice-Evans C. Packer L (CRC, Boca Raton, Fla.), pp 329-394 The flavonoidluteolin (3′,4′,5,7-tetrahydroxyflavone), abundant in celery, greenpepper, parsley, perilla leaf, and chamomile tea (Shimoi K, et al.(1998) Intestinal absorption of luteolin and luteolin 7-O-β-glucoside inrats and humans. FEBS Lett 438:220-224), is of particular interest formodulating immune reactions as several studies comparing theanti-inflammatory properties of luteolin with other flavonoids such asquercetin, genistein, or hesperetin in peripheral macrophages foundluteolin to be most potent (Xagorari A, et al. (2001) Luteolin inhibitsare endotoxin-stimulated phosphorylation cascade and proinflammatorycytokine production in macrophages. Pharmacol Exp Ther 296:181-187;Comalada M, et al (2006) Inhibition of proinflammatory markers inprimary hone marrow-derived mouse macrophages by naturally occurringflavonoids: Analysis of the structure-activity relationship. BiochemPharmacol 72: 1010-1021).

Luteolin has been shown to inhibit LPS-induced NF-κB transcriptionalactivity in intestinal epithelial cells, mouse bone-marrow deriveddendritic cells (Kim J S, Jobin C (2005) The flavonoid luteolin preventslipopolysaccharide-induced NF-κB signaling and gene expression byblocking IκB kinase activity in intestinal epithelial cells andbone-marrow derived dendritic cells. Immunology 115:375-387), murinemacrophage cells, and rat fibroblasts (Kim S H, et al (2003) Luteolininhibits the nuclear factor-κB transcriptional activity in Rat-1fibroblasts. Biochem. Pharmacol 66:955-963). In another study, luteolininhibited LPS-stimulated TNF-α and IL-6 in a murine macrophage cellline. These studies suggest luteolin modulates cell signaling pathwaysactivated by LPS and subsequent production of inflammatory cytokines.

Diosmin containing supplements have been in use in Europe for the pastthree decades. Diosmin is currently considered a vascular-protectingagent and has been used safely for treatment of chronic venousinsufficiency/varicose veins, hemorrhoids, lymphedema, and diabetes (LeLyseng-Williamson and Perry 2003; Mlakar and Kosorok 2005; Nicolaides2005). Clinical trials have used doses of 500-2,000 mg per day orallyfor up to one year. Throughout these trials, diosmin demonstrated anexcellent safety profile and were well tolerated. Adverse events withsuch complexes were rare; and when they occurred, they were always mild,and transient. The side effects typically observed were mild cases ofdigestive intolerance requiring no changes in treatment.

Pharmacokinetic studies demonstrated diosmin is rapidly transformed inthe intestine to diosmetin, its aglycone form (Cova et al., 1992; Labrid1994; Meyer 1994). Diosmetin is subsequently absorbed and distributedthroughout the body with a plasma half-life of 26-43 hrs. Data from thisstudy was used to help determine the shape of the dose response, andoptimal dose for human clinical trials to test diosmin as an effectiveanti-Stat3 prenatal supplement in infected, or potentially infectedpregnant mothers. Diosmin can be used as a prenatal supplement to defendagainst autism risk, just as folate is currently used as prophylaxisagainst neural tube defects in human offspring.

Park et al., 2008 found that EGCG inhibits STAT3 activation as anintegral part of inhibition of keloid formation. In an earlier study itwas found that silibinin, a flavonoid, inhibits constitutive activationof STAT3, and causes caspase activation and apoptotic death of humanprostate carcinoma cells (Agarwal et al., 2007). Prior to this, it wasdemonstrated that in vivo treatment of SJL/J mice with quercetin, aflavonoid, (i.p. 50 or 100 μg every other day which is equal to 1.25mg/kg/day or 2.5 mg/kg/day) ameliorates experimental autoimmuneencephalitis (EAE) by inhibiting IL-12 production and neuralantigen-specific Th1 differentiation. In vitro treatment of activated Tcells with this same flavonoid quartering blocks IL-12-induced tyrosinephosphorylation of JAK2, TYK2, STAT3, and STAT4, yielding a reducedIL-12-induced T cell proliferation and Th1 differentiation (Muthian andBright 2004).

Our studies indicated that IL-6 activates the JAK2/STAT3 pathway, as N2aneuronal cells and brain homogenates from newborn IL-6-induced MIA(IL-6/MIA) offspring showed increased neuronal JAK2/STAT3phosphorylation. In adulthood, these mice showed deficits in socialinteraction, suggesting that not only does IL-6 activate the JAK2/STAT3pathway, but that it is also involved in the abnormal behavioralpathologies observed in MIA offspring and autism and related disorders.Next we investigated if inhibition of JAK2/STAT3 signaling couldattenuate MIA-induced pathologies. Previous research by our laboratoryhas shown that bioflavonoids such as epi-gallocatechin gallate (EGCG) orluteolin, inhibit IFN-γ induced STAT1 activation and attenuateproduction of pro-inflammatory cytokines in cultured and primarymicroglial cells (Giunta et al., 2006, Jagtap et al., 2009 andRezai-Zadeh et al., 2008).

We investigated the prophylactic effects of two flavonoids which possessbetter bioavailability and safety than these previously testedcompounds. These two flavonoids are, luteolin, and its structuralanalog, diosmin. We found that JAK2/STAT3 phosphorylation and signalingas well as behavioral abnormalities in IL-6 induced MIA offspring couldbe ameliorated with these naturally occurring compounds. Our resultsshowed that administration of diosmin (10 mg/kg/day) was able to blockthe STAT3 signal pathway; significantly opposing IL-6-induced abnormalbehavior and neuropathological abnormalities in MIA/adult offspring.Using guidelines put forth by the Food and Drug Administration(Reagan-Shaw et al., 2008), this 10 mg/kg/day dose in mice is equivalentto 0.81 mg/kg/day in humans which translates into 48.6 mg/day for a 60kg person.

The present invention includes methods for the treatment of autoimmunedisorders such as autism, schizophrenia, and diabetes through theadministration of flavonoids. Flavonoids, luteolin, diosmin, and itsaglycone form, diosmetin, were found to inhibitactivation/phosphorylation of STAT3 induced by IL-6 in cultured neuronalcells. These flavonoids have a profound and dose-dependent effect oninhibiting STAT3 activation using PC12 and N2a cells as evidenced bymarkedly decreased STAT3 phosphorylation by Western Blot Analysis. Thiseffect is mediated by a reduction in downstream productions of the STAT3signal pathway. Pregnant mice (E4) were treated with diosmin in thepresence or absence of mouse IL-6 and autistic phenotypes, includingpathology and behavioral changes in these treated mice, and werecompared with a control. It was found that diosmin attenuatesIL-6-induced autistic phenotype in mice. Due to the nature of thesecompounds, they are also effective in treating other diseases includingschizophrenia and diabetes.

SUMMARY OF THE INVENTION

An illustrative embodiment of the present invention includes a method oftreating an autoimmune disorder such as autism, schizophrenia, ordiabetes through the administration of a therapeutically effectiveamount of a flavonoid. The flavonoid is preferably luteolin, diosmin, ordiosmetin or any of their structural analogues or derivatives.

The flavonoid is preferably administered orally. The flavonoid can beadministered at 25 mg, 50 mg, 100 mg, 200 mg, 400 mg, 600 mg, 800 mg,1000 mg, 1200 mg, 1400 mg, 1600 mg, 1800 mg, and 2000 mg. The maximumdosage can be 2000 mg. The dosage can be weight dependent. The dosageadministered can be between 0.81 mg/kg/day and 2.5 mg/kg/day. Thepreferred dose of the flavonoid is at least 0.81 mg/kg/day.

In another embodiment, the present invention includes a method oftreating autism through the administration of a therapeuticallyeffective amount of a flavonoid such as luteolin, diosmin, or diosmetin.

An additional embodiment includes a method of reducing inflammation dueto an autoimmune disease such as autism, schizophrenia, or diabetesthrough the administration of a flavonoid. The flavonoid may beluteolin, diosmin, or diosmetin. Preferably the flavonoid isadministered orally and the preferred dose is at least 0.81 mg/kg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1A is an image of a Western Blot Analysis showing that IL-6 inducesJak2 phosphorylation in cultured neuron-like cells (N2a cells). CulturedN2a cells were treated with 50 ng/mL of IL-6 for a range of time points.Cell lysates were prepared and subjected to Western Blot Analysis.Densitometry analysis shows the ratio of phosphor-Jak2 to the totalJak2. One way ANOVA showed that IL-6 significantly activates Jak2 in atime-dependent manner (**P<0.005).

FIG. 1B is an image of a Western Blot Analysis showing that the presenceof luteolin significantly inhibits IL-6-induced Jak2 phosphorylation(*P<0.01). Cultured Na2 cells were co-treated with IL-6 (50 ng/mL) andluteolin at various doses for 30 minutes. Cell lysates were prepared andsubjected to Western Blot Analysis. Densitometry analysis reveals theratio of phosphor-Jak2 to total Jak2.

FIG. 1C is an image of a Western Blot Analysis showing that IL-6 inducesSTAT3 phosphorylation in cultured neuron-like cells (N2a cells).Cultured N2a cells were treated with 50 ng/mL of IL-6 for a range oftime points. Cell lysates were prepared and subjected to Western BlotAnalysis. Densitometry analysis shows the ratio of phosphor-STAT3 to thetotal STAT3. One way ANOVA showed that IL-6 significantly activatesSTAT3 in a time-dependent manner (**P<0.005).

FIG. 1D is an image of a Western Blot Analysis showing that the presenceof luteolin significantly inhibits IL-6-induced STAT3 phosphorylation(*P<0.01). Cultured Na2 cells were co-treated with IL-6 (50 ng/mL) andluteolin at various doses for 30 minutes. Cell lysates were prepared andsubjected to Western Blot Analysis. Densitometry analysis reveals theratio of phosphor-STAT3 to total STAT3. Data is representative of threeindependent experiments. Similar results were obtained in murine primarycultured neuronal cells using antibody specifically againstphosphor-JAK2/STAT3 (Ser⁷²⁷) and in N2a cells using antibodyspecifically against phosphor-STAT3 (Ser⁷⁰⁵).

FIG. 2A is an image of a Western Blot Analysis showing both STAT3inhibitor 531-201 and diosmin (a glycoside of a structurally similarflavonoid to luteolin) reduce Jak2 phosphorylation. Brain homogenateswere prepared from newborn mice from mothers injected with IL-6,IL-6/S31-201, IL-6/diosmin, or PBS (control) and subjected to WesternBlot Analysis and cytokine ELISA. Densitometry analysis shows the ratioof phospho-Jak2 to total Jak2. One way ANOVA showed that both S31-201and diosmin significantly inhibit Jak2 signaling (P<0.005).

FIG. 2B is an image of a Western Blot Analysis showing both STAT3inhibitor S31-201 and diosmin (a glycoside of a structurally similarflavonoid to luteolin) reduce STAT3 phosphorylation. Brain homogenateswere prepared from newborn mice from mothers injected with IL-6,IL-6/S31-201, IL-6/diosmin, or PBS (control) and subjected to WesternBlot Analysis and cytokine ELISA. Densitometry analysis shows the ratioof phospho-STAT3 to total STAT3. One way ANOVA showed that both S31-201and diosmin significantly inhibit STAT3 signaling (P<0.005).

FIG. 3 is a graph showing a significant reduction of TNF- and IL1cytokines in brain homogenates from IL-6/S31-201 and IL-6/diosminnewborn mice when compared to IL-6 only (MIA model) newborn mice(**P<0.01). Pro-inflammatory cytokine analysis by ELISA was conducted onnewborn mouse brain homogenates. Data are represented as mean±SD of eachcytokine in brain homogenates (pg/mg total protein) from newborn mice.

FIG. 4A is a graph showing that maternally blocking the STAT3 signalpathway with diosmin opposes IL-6-induced abnormal behavior in MIA/adultoffspring as shown by the results of the open-field test. In theopen-field test, offspring of mice treated with either S31-201 ordiosmin enter the center more often than those treated only with IL-6(**P<0.01) and are nearly similar to control mice. Offspring of miceintraperitoneally (i.p.) treated with IL-6 (5 g/mouse) in the absence orpresence of STAT3 inhibitor S31-201 (4 g/mouse; i.p.) or with diosmin(10 mg/kg/day; oral administration).

FIG. 4B is a graph showing that maternally blocking the STAT3 signalpathway with diosmin opposes IL-6-induced abnormal behavior in MIA/adultoffspring as shown by the results of the social interaction test. In thesocial interaction test, the social chamber is defined as (thepercentage of time in the social chamber)−(the percentage of time in theopposite chamber). Control mice reveal a strong preference for thesocial chamber. Offspring of mice treated with either S31-201 or diosminalso enter the social chamber more often than those treated only withIL-6 (**P<0.005) and are nearly similar to control mice. Offspring ofmice intraperitoneally (i.p.) treated with IL-6 (5 g/mouse) in theabsence or presence of STAT3 inhibitor S31-201 (4 g/mouse; i.p.) or withdiosmin (10 mg/kg/day; oral administration).

FIG. 5 is a graph showing that diosmin reduces pro-inflammatorycytokines Brain homogenates were prepared from offspring of mice treatedwith IL-6, IL-6/S31-201, IL-6/diosmin or PBS (control). Pro-inflammatorycytokine analysis by ELISA was conducted on the mouse brain homogenates.Data are represented as mean±SD of each cytokine in brain homogenates(pg/mg total protein) from these mice. Analysis of results revealed asignificant reduction of TNF- and IL1 cytokines in brain homogenatesfrom IL-6/S31-201 and IL-6/diosmin adult offspring when compared to MIAonly mothers (IL-6 treatment only) (**P<0.05).

FIG. 6A is an image of a Western Blot Analysis showing a notablereduction in STAT3 phosphorylation in brain homogenates fromIL-6/S31-201 and IL-6/diosmin adult offspring when compared to theoffspring of MIA-only mothers (IL-6 treatment only). Western BlotAnalysis was conducted with antibodies specifically againstphosphor-STAT3 (Ser⁷²⁷) and total STAT3.

FIG. 6B is a graph showing the results of densitometry analysisrevealing the ratio of phosphor-STAT3 to total STAT3. Analysis ofresults showed a significant reduction of STAT3 phosphorylation fromIL-6/S31-201 and IL-6/diosmin adult offspring when compared to offspringof MIA-only (IL-6 treatment only) mothers (**P<0.005).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

Throughout this application various publications are referenced.Disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The “therapeutically effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. Atherapeutically effective amount of the flavonoids such as luteolin,diosmin, or diosmetin, their analogues or any combination thereof isthat amount necessary to provide a therapeutically effective result invivo. A therapeutically effective result refers to a dose of theflavonoid that is sufficient to provide a physical benefit or atherapeutic benefit. The amount of flavonoids such as luteolin, diosmin,or diosmetin or any combination thereof must be effective to achieve aresponse, including but not limited to total prevention of (e.g.,protection against) and to improved survival rate or more rapidrecovery, or improvement or elimination of symptoms associated withinflammatory disorders, autoimmune disorders, or other indicators as areselected as appropriate measures by those skilled in the art. Inaccordance with the present invention, a suitable single dose size is adose that is capable of preventing or alleviating (reducing oreliminating) a symptom in a patient when administered one or more timesover a suitable time period. One of skill in the art can readilydetermine appropriate single dose sizes for systemic administrationbased on the size of a mammal and the route of administration.

The term “administration” or “administering” as used throughout thespecification to describe the process in which a flavonoid is deliveredto a patient for treatment purposes. This includes parenternal;intraventricular; intraparenchymal; including spinal cord and brainstem; intracisternal; intrastriatal; intranigral; orally; rectally; andother routes that allow the flavonoid to contact cells. The term“parenternal” refers to subcutaneous, intracutaneous, intravenous,intramuscular, intrathecal, intralesional, intraartricular,intraarterial, intrasynovial, intrasternal, intrathecal, andintracranial injection, as well as various infusion techniques. Theflavonoid may be administered independently or in combination with othercompounds. Administration will often depend on the disease and level ofprogression.

The term “autoimmune disorder” as used throughout refers to thosedisease states and conditions wherein the immune response of the patientis directed against the patient's own constituents, resulting in anundesirable and often terribly debilitating condition. As used herein,“autoimmune disorder” is intended to further include autoimmuneconditions, syndromes, and the like.

The term “flavonoid” is used throughout the specification to identifythose polyphenolic compounds that are ubiquitous in nature. The term“flavonoid” as used herein includes isoflavones, anthocyanidins,flavans, flavonols, flavones, citrus flavonoids, hesperidin, chalcones,catechins, rutin, and flavanones.

We have found that the flavonoids, luteolin and diosmin/diosmetin, havea profound and dose-dependent effect on inhibiting STAT3 activationusing PC12 and N2a cells, as evidenced by markedly decreased STAT3phosphorylation on a Western blot analysis. Most importantly, thiseffect is mediated by a reduction in downstream productions of STAT3signal pathway.

We have shown that the citrus bioflavonoid luteolin inhibits Stat3phosphorylation in both murine neuron-like cells and primary neuronalcells challenged with IL-6. Diosmin (a glycoside of a structurallysimilar flavanoid to luteolin), administered orally, significantlychanges behavioral deficits in social interaction in IL-6/MIA adultoffspring. Additionally, diosmin reduces the CNS levels inpro-inflammatory cytokines consistent with Jak2/Stat3 signal pathwayinhibition.

Preliminary studies showed that the citrus bioflavonoid, luteolin,inhibits neuronal Jak2/Stat3 phosphorylation in both murine neuron-like(N2a) cells and primary cultured neuronal cells challenged with IL-6. Asa validation of these findings, the effects of a Stat3 inhibitor(S31-201) and diosmin, a flavanoid structurally similar to luteolin, wasexamined on Jak2/Stat3 signaling. When either agent was applied topregnant mice with an injection of IL-6 to induce MIA, Jak2/Stat3phosphorylation and pro-inflammatory cytokines were both significantlyreduced in brain homogenates from newborn mice. It was also found thatdiosmin administered orally in chow (10 mg/kg/day) significantly changesbehavioral deficits in social interaction and reduces pro-inflammatorycytokines in brain tissues of IL-6/MIA adult offspring. Finally, it wasdemonstrated that diosmin reduces CNS levels of pro-inflammatorycytokines consistent with Jak2/Stat3 signal pathway inhibition.

Methods Reagents

Luteolin (>95% purity by HPLC) was purchased from Sigma (St Louis, Mo.,USA). Diosmin (>90% purity by HPLC) was purchased from Axxora (SanDiego, Calif., USA). Antibodies against JAK2, phospho-JAK2, STAT3 andphospho-STAT3 were obtained from Cell Signaling Technology (Danvers,Mass., USA). ELISA kits for tumor necrosis factor-α(TNF-α) andInterleukin-1β (IL-1β) were obtained from R&D Systems (Minneapolis,Minn., USA). BCA protein assay kit was purchased from PierceBiotechnology (Rockford, Ill., USA). Murine recombinant IL-6 waspurchased from eBioscience (San Diego, Calif., USA).

Primary Cell Culture

Cerebral cortices were isolated from C57BL/6 mouse embryos, between 15and 17 days in utero. After 15 min of incubation in trypsin (0.25%) at37° C., individual cortices were mechanically dissociated. Cells werecollected after centrifugation at 1200 rpm, resuspended in DMEMsupplemented with 10% fetal calf serum, 10% horse serum, uridine (33.6g/mL; Sigma) and fluorodeoxyuridine (13.6 g/mL; Sigma), and plated in 24well collagen coated culture plates at a density of 2.5×10⁵ cells perwell.

N2a (murine neuroblastoma) cells, purchased from the American TypeCulture Collection (ATCC, Manassas, Va.) were grown in complete EMEMsupplemented with 10% fetal calf serum. Cells were plated in 24 wellcollagen coating culture plates at a density of 1×10⁵ cells per well.After overnight incubation, N2a cells were incubated in neurobasal mediasupplemented with 3 mM dibutyryl cAMP in preparation for treatment.

Cells were treated with 50 ng/mL murine recombinant IL-6 for a range oftime points (0, 15, 30, 45, 60 or 75 min) in the presence or absence ofvarious concentrations of luteolin (0, 1.25, 2.5, 5, 10, 20 μM) for 30min.

Mice

Pregnant C57BL/6 mice, embryonic day 2 (E2) were obtained from JacksonLaboratory (Bar Harbor, Mass.) and individually housed and maintained inan animal facility of the University of South Florida (USF). Allsubsequent experiments were performed in compliance with protocolsapproved by the USF Institutional Animal Care and Use Committee. AtE12.5, mice were intraperitoneally (i.p.) challenged (one time only)with murine recombinant IL-6 (5 μg dissolved in 200 μL, of PBS/mouse) inthe presence or absence of the STAT3 inhibitor S31-201 (4 μg dissolvedin 200 μL, of PBS/mouse) and/or treated with diosmin, administeredorally (10 mg/kg/day, 0.005% in NIH31 chow). Intraperitoneal PBSinjection (200 μL) was used as the control for IL-6 administration.

Western Blot

Cultured cells were lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5,150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodiumpyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin,1 mM PMSF) as described previously (Tan et al. 2002). Mouse brains wereisolated under sterile conditions on ice and placed in ice-cold lysisbuffer. Brains were then sonicated on ice for approximately 3 min,allowed to stand for 15 min at 4° C., then centrifuged at 15,000 rpm for15 min at 4° C. Total protein content was estimated using the BCAprotein assay (Pierce Biotechnology) and aliquots corresponding to 100μg of total protein were electrophoretically separated using 10% Trisgels. Electrophoresed proteins were then transferred to nitrocellulosemembranes (Bio-Rad, Richmond, Calif.), washed in Tris buffered salinewith 0.1% Tween-20 (TBS/T), and blocked for 1 h at ambient temperaturein TBS/T containing 5% (w/v) non-fat dry milk. After blocking, membraneswere hybridized overnight at 4° C. with various primary antibodies.Membranes were then washed 3× for 5 min each in TBS/T and incubated for1 h at ambient temperature with the appropriate HRP-conjugated secondaryantibody (1:1000, Pierce Biotechnology). Primary antibodies were dilutedin TBS/T containing 5% BSA and secondary antibodies in TBS containing 5%(w/v) of non-fat dry milk. Blots were developed using the luminolreagent (Pierce Biotechnology). Densitometric analysis was conductedusing a FluorS Multiimager with Quantity One™ software (Bio-Rad,Hercules, Calif.). For phospho-STAT3 detection, membranes were probedwith a phospho-Ser727 STAT3 antibody (1:1000) and stripped withstripping solution and then re-probed with antibody that recognizestotal STAT3 (1:1000). Similarly for phospho-JAK2 detection, membraneswere probed with phospho-JAK2 (1:1000) and stripped and re-probed fortotal JAK2 (1:1000).

ELISA Cytokine Analysis

Mouse brain homogenates were prepared as described above and used at adilution of 1:10 in PBS for these assays. Brain tissue-solubilizedcytokines were quantified using commercially available ELISAs that allowfor detection of IL-1β, and TNF-α. Cytokine detection was carried outaccording to the manufacturer's instruction. Total protein content wasdetermined as described above and data represented as pg of cytokine/mgtotal cellular protein for each cytokine

Behavioral Testing

Open field (OF)—The OF behavioral analysis is a test of both locomotoractivity and emotionality in rodents (Radyushkin et al. 2009). Mice wereplaced in a 50×50 cm white Plexiglas box brightly lit by fluorescentroom lighting and six 60 W incandescent bulbs approximately 1.5 m abovethe box. Activity was recorded by a ceiling-mounted video camera andanalyzed from digital video files either by the automated trackingcapabilities of Ethovision or counted using the behavior tracker(version 1.5, www.behaviortracker.com), a software-based event-recorder.The total distance moved and numbers of entries into the center of thearena (central 17 cm square) were determined in a 10 minute session.

Social interaction (SI)—The testing apparatus consisted of a 60×40 cmPlexiglas box divided into three chambers. Mice were able to movebetween chambers through a small opening (6×6 cm) in the dividers.Plastic cylinders in each of the two side chambers contained the probemice, and numerous 1 cm holes in the cylinders enabled test and probemice to contact each other. Test mice were placed in the center chamber,with an overhead camera recording their movements. Mice were allowed 5min of exploration time in the box, after which an unfamiliar, same-sexprobe mouse from the same experimental group was placed in one of tworestraining cylinders (Radyushkin et al. 2009). The Ethovision software(Noldus, Leesburg, Va.) program measured time spent in each of the threechambers, and social preference was defined as follows: (% time spent inthe social chamber)−(% time spent in the opposite chamber).

Statistical Analysis

All data were normally distributed; therefore, in instances of singlemean comparisons, Levene's test for equality of variances followed byt-test for independent samples was used to assess significance. Ininstances of multiple mean comparisons, analysis of variance (ANOVA) wasused, followed by post hoc comparison using Bonferroni's method. Alphalevels were set at 0.05 for all analyses. The STATistical package forthe social sciences release 10.0.5 (SPSS Inc., Chicago, Ill., USA) wasused for all data analysis.

Results Luteolin Inhibits IL-6 Induced Neuronal JAK2/STAT3Phosphorylation

To confirm the role of IL-6 in regulating the JAK2/STAT3 pathway, wetreated murine neuron-like (N2a) cells and primary cultured neuronalcells with 50 ng/mL of murine recombinant IL-6 in a time-dependentmanner. Western blot analysis of cell lysates showed that IL-6 treatmentleads to a time-dependent increase in JAK2 (FIG. 1A) and STAT3 (FIG. 1C)phosphorylation. Densitometric analysis indicated significant and steadyincreases in JAK2 phosphorylation (**P<0.005) beginning at 30 min andcontinuing until 75 min when the timed analysis was concluded (FIG. 1A).Densitometric analysis of STAT3 phosphorylation (**P<0.005) showed asignificant and maximal increase at 30 min, with no further significantincreases at subsequent time points.

We next examined the effects of luteolin on IL-6 induced JAK2/STAT3phosphorylation. Murine N2a cells and primary cultured neuronal cellswere again challenged with 50 ng/mL murine recombinant IL-6 andco-treated with increasing concentrations of luteolin (0-20 μM) for 30min. Following Western blot analysis of cell lysates, we found thatluteolin reduces IL-6 induced JAK2 phosphorylation (FIG. 1B) and STAT3phosphorylation (Ser⁷²⁷) (FIG. 1D) in both murine N2a and primaryneurons in a dose dependent manner with significant reductions beginningat 10 μM. Densitometric analysis showed that luteolin inhibited JAK2 andSTAT3 (Ser⁷²⁷) phosphorylation by almost 50% (*P<0.01 and **P<0.001,respectively). It is important to note that luteolin did not affectapoprotein levels of JAK2 or STAT3. The data shown is representative ofthree independent experiments. Similar results were obtained in murineprimary cultured neuronal cells using antibody specifically againstphospho-Jak2/Stat3 (Ser⁷²⁷) and in N2a cells using antibody specificallyagainst phospho-Stat3 (Ser⁷⁰⁵).

Both STAT3 inhibitor, S31-201, and diosmin reduce JAK2/STAT3phosphorylation and pro-inflammatory cytokine production in the braintissues of IL-6/MIA newborn mice

We subsequently extended the in vitro results to an animal model ofMIA-induced autism by examining the effects of STAT3 inhibitor (S31-201)and diosmin (a flavonoid structural analog of luteolin), on JAK2/STAT3phosphorylation and signaling. When either agent was co-administered topregnant mice intraperitoneally with IL-6, JAK2/STAT3 phosphorylationand pro-inflammatory cytokine levels were both significantly reduced inthe brain homogenates of newborn mice. Western blot analysis of brainhomogenates shows that both S31-201 and diosmin significantly reduceIL-6 induced JAK2 (FIG. 2A) and STAT3 (FIG. 2B) phosphorylation(*P<0.005).

As shown in FIG. 2, both STAT3 inhibitor (S31-201) and diosmin (aglycoside of a structurally similar flavonoid to luteolin) reduceJAK2/STAT3 phosphorylation. Brain homogenates were prepared from newbornmice from mothers injected with IL-6, IL-6/S31-201, IL-6/diosmin or PBS(control) (n=6, 3 female/3 male) and subjected to Western blot analysisand cytokine ELISA. Most notably, the treatment of S31-201 or diosminsignificantly inhibits IL-6-induced JAK2/STAT3 phosphorylation in braintissues from newborn mice. Densitometry analysis shows the ratio ofphospho-JAK2/STAT3 to total JAK2/STAT3 as indicated below the figures.One-way ANOVA showed that both significantly inhibit JAK2/STAT3signaling (P<0.005).

Pro-inflammatory cytokine ELISA showed significant increases in TNF-αand IL-1β levels in the brain homogenates of new born mice from IL-6treated dams compared to those of control dams (FIG. 3). These increaseswere significantly reduced by almost 50% in the presence of S31-201 ordiosmin (**P<0.01) (FIG. 3).

As shown in FIG. 3, STAT3 inhibitor (S31-201) and diosmin reducepro-inflammatory cytokines in the brain tissues of IL-6/MIA/newbornmice. Pro-inflammatory cytokine analysis by ELISA was conducted on thesenewborn mouse brain homogenates. Data are represented as mean±SD of eachcytokine in brain homogenates (pg/mg total protein) from these newbornmice. Analysis of results revealed a significant reduction of TNF-α andIL-1β cytokines in brain homogenates from IL-6/S31-201 andIL-6/Diosmin/newborn mice when compared to IL-6 only (MIA model) newbornmice (**P<0.01).

Maternally blocking the STAT3 signal pathway with the STAT3 inhibitor,S31-201 or diosmin opposes IL-6-induced abnormal behavior in MIA/adultoffspring

We next determined whether diosmin would attenuate behavioralabnormalities observed in the adult offspring of IL-6 treated dams.Pregnant mice were treated one time with 5 μg/mL IL-6 in the presence orabsence of S31-201 (4 μg/mL) or diosmin (10 mg/kg/day diosmin)administered orally in chow. We also treated control mice (non-IL-6treated) with the STAT3 inhibitor, S31.

The adult offspring of these mice were examined for behavioral outcomesusing the open field and social interaction tests to examine anxiety andsocial interaction, respectively. Our results demonstrate that S31-201or diosmin co-treatment significantly attenuates the behavioral deficitsseen in the adult offspring of IL-6 treated animals. In the open-fieldtest, offspring of mice treated with either S31-201 or diosmin showedbehaviors comparable to that of control mice, entering the center moreoften than IL-6 treated animals (**P<0.01) (FIG. 4A). In addition, anANOVA on time spent in the inner section showed a significant maineffect of group (P<0.05) and LSD post hoc tests showed that the IL-6/MIAmice spend less time in the inner section compared to S31-201 or diosmintreated mice (P<0.05). As these could be due to simply increasedlocomotion in one group, ANOVA on distance traveled in the inner sectionwas performed and did not indicate a main effect (P=0.069). Analyses ofdistance traveled in the outer section did not reveal a significantdifference or a statistical trend towards a significant difference(P>0.15) among groups. We did not find a significance betweenPBS-treated mice and S31-treated control (non-IL-6 injected) mice(P>0.05 with n=5).

As shown in FIG. 4, maternally blocking STAT3 signal pathway withdiosmin opposes IL-6-induced abnormal behavior in MIA/adult offspring.Offspring of mice (n=8, 4 female/4 male) intraperitoneally (i.p.)treated with IL-6 (5 μg/mouse) in the absence or presence of STAT3inhibitor (S31-201; 4 μg/mouse; i.p.) (Siddiquee et al. 2007) or withdiosmin [oral administration; (10 mg/kg/day)]. FIG. 4A shows that in theopen-field test, offspring of mice treated with either S31-201 ordiosmin enter the center more often than IL-6 (**P<0.01) and are nearlysimilar to control mice. Heightened anxiety and social interactiondeficits are hallmarks of schizophrenia and autism. Reluctance to enterthe center portion of a well-lit open field is usually taken as ameasure of heightened anxiety under mildly stressful conditions (Smithet al., 2007). FIG. 4B shows that in the social interaction test, aspreviously reported, the social chamber was defined as (percentage oftime in social chamber)−(percentage of time in opposite chamber). Mostnotably, control mice reveal a strong preference for the social chamber.Interestingly, the social impairment of offspring is significantlyimproved by maternal administration of STAT3 inhibitor, S31-201 ordiosmin (**P<0.005).

In the social interaction test, the adult offspring of control mice showa strong preference for the social chamber almost double that of theadult offspring of IL-6 treated mice. To quantify social interaction,exploration demonstrated as sniffing time was analyzed via ANOVA. Therewas a main effect of group (P<0.05); the S31-201 or diosmin miceexhibited an increase in sniffing compared to IL-6/MIA mice (P<0.05). Wedid not observe any social grooming, chasing, dominant mounts, pinning,boxing, or biting. There were no significant differences in total movetime (P>0.07) and total distance traveled (P>0.05).

This social impairment observed in IL-6 adult offspring wassignificantly attenuated by maternal co-treatment with S31-201 ordiosmin as these mice show a preference for the social chambercomparable to that of control adult offspring (**P<0.005) (FIG. 4B).Considering the above data, it can be seen that IL-6 induced JAK2/STAT3phosphorylation plays an essential role in precipitating behavioralabnormalities seen in the adult offspring of IL-6 treated dams andregulation of this pathway by diosmin can attenuate these behavioralabnormalities.

Diosmin reduces pro-inflammatory cytokines and STAT3 phosphorylation inIL-/MIA adult offspring

After behavioral testing, adult offspring were sacrificed to confirmthat inhibition of STAT3 phosphorylation by diosmin attenuatesIL-6/JAK2/STAT3 induced behavioral abnormalities. At sacrifice, brainhomogenates were prepared from offspring of control mice and micetreated with IL-6, IL-6/S31-201, and IL-6/Diosmin. Pro-inflammatorycytokine ELISA showed significant increase in TNF-α and IL-1β levels inthe homogenates of IL-6 adult offspring. Maternal co-treatment withS31-201 or diosmin significantly reduces TNF-α and IL-1β cytokine levelssignificantly, with diosmin showing slightly more significant reductionsas shown in FIG. 5 (**P<0.05).

As shown in FIG. 5, Diosmin reduces pro-inflammatory cytokines inIL-6/MIA adult offspring. At sacrifice, brain homogenates were preparedfrom offspring of mice treated with IL-6, IL-6/S31-201, IL-6/Diosmin orPBS (control) (n=8, 4 female/4 male). Pro-inflammatory cytokine analysisby ELISA was conducted on these mouse brain homogenates. Data arerepresented as mean±SD of each cytokine in brain homogenates (pg/mgtotal protein) from these mice. Analysis of results revealed asignificant reduction of TNF-α and IL-1β cytokines in brain homogenatesfrom IL-6/31-201 and IL-6/Diosmin adult offspring when compared tooffspring of MIA only mothers (IL-6 treatment only) (**P<0.05).

Western blot analysis of phospho- and total STAT3 shows that IL-6treatment of dams increases STAT3 phosphorylation in the brainhomogenates of adult offspring while co-treatment with either S31-201 ordiosmin leads to a significant reduction of STAT3 phosphorylation(**P<0.005) (FIG. 6).

As shown in FIG. 6, Diosmin reduces STAT3 phosphorylation in IL-6/MIAadult offspring. FIG. 6A shows a Western blot analysis with antibodiesspecifically against phospho-STAT3 (Ser⁷²⁷) and total STAT3 shows anotable reduction of STAT3 phosphorylation in brain homogenates fromIL-6/S31-201 and IL-6/Diosmin/adult offspring when compared to offspringof MIA only mothers (IL-6 treatment only). The densitometric analysisshown in FIG. 6B reveals the ratio of phospho-STAT3 to total STAT3 asindicated below the figure. In support, analysis of results showed asignificant reduction of STAT3 phosphorylation from IL-6/S31-201 andIL-6/Diosmin/adult offspring when compared to offspring of MIA onlymothers (IL-6 treatment only) (**P<0.005).

Elucidating the mechanisms and pathways involved in neurodevelopmentaldisorders such as autism and schizophrenia is important in not onlyunderstanding the etiology of these disorders but also to discover earlydiagnostic markers, and prophylactic therapies, in addition totherapeutic strategies to attenuate the associated symptoms. Previousresearch by Smith and colleagues has supported the role of exogenousIL-6 in precipitating the behavioral deficits and increases inpro-inflammatory cytokine release seen in MIA offspring, and potentiallyautistic and schizophrenic individuals, in addition to demonstratingthat its inhibition can attenuate these pathologies (Smith et al. 2007).We further examined the role of IL-6 in MIA by characterizing the roleof JAK2/STAT3 phosphorylation in precipitating behavioral andpathological outcomes. We also determined if inhibition of thisphosphorylation had the ability to attenuate these behavioral deficitsand/or pathologies observed in the adult offspring of IL-6/MIA mice.Previous research by our laboratory has shown that bioflavonoidsregulate STAT1 phosphorylation, as we showed that luteolin (Rezai-Zadehet al. 2008) or EGCG (Giunta et al. 2006) inhibited IFN-γ induced STAT1phosphorylation. We examined if diosmin, a luteolin analog with betterbioavailability, had similar effects, inhibiting IL-6 induced STAT3phosphorylation.

We confirmed the role of IL-6 induced JAK2/STAT3 phosphorylation inprecipitating pathological and behavioral deficits seen previously inIL-6/MIA animal models. The results of our study demonstrate that IL-6induces JAK2/STAT3 phosphorylation resulting in the release ofpro-inflammatory cytokines both in vitro and in vivo. Western blotanalyses of cell lysates from murine N2a cells and primary culturedneuronal cells showed time-dependent increases in JAK2/STAT3phosphorylation following IL-6 treatment. Analysis of brain homogenatesof newborn mice from IL-6 treated dams similarly showed increases inJAK2/STAT3 phosphorylation, in addition to increases in releasedpro-inflammatory cytokines TNF-α and IL-1β.

We measured IL-6 levels in brain homogenates from the offspring usingELISA. The level of IL-6 was undetectable which is in contrast to theresults previously reported by Samuelsson et al. 2006. As previouslymentioned, this group found that IL-6 mRNA levels remain elevated in thehippocampi of the offspring at 4 and 24 weeks of age; indicative of theongoing state of immune dysregulation in adult autistic brains. We thussuggest IL-6 itself may not be a contributory factor for the in vivochronic inflammatory pathogenic affects, but rather for the in uteroeffects. Therefore measuring IL-6 in the offspring may not berepresentative of its exposure in utero. Rather, the indirect effects ofIL-6 pro-inflammatory activation in utero of the JAK2/STAT3 pathway, asevidenced by increased TNF-α and IL-1β production, may be at play in anautism mouse phenotype. Thus, with evidence that JAK2/STAT3phosphorylation induced pathological outcomes in the offspring of IL-6treated animals, we examined whether or not this phosphorylationcontributed to the behavioral deficits observed in the offspring of IL-6treated dams.

There is much epidemiologic evidence indicating that environmentalcontributions, including prenatal infections which can lead to MIA, maylead to the genesis of autism (Arndt et al. 2005 and Libbey et al 2005).Previous animal models, developed by Fatemi and Folsom 2009 and others,have shown that immune challenges during pregnancy lead to abnormalbrain structure and function in the exposed offspring that replicateabnormalities observed in brains of subjects with autism (Fatemi et al.2005, Fatemi et al. 2008, Meyer et al. 2006, Meyer et al. 2007 and Shiet al. 2005). Abnormal CNS changes in the offspring following infectionat E9, which corresponds to infection at the middle of the firsttrimester (Fatemi et al. 2005 and Shi et al. 2003), and E18, whichcorresponds to infection in late second trimester (Fatemi et al. 2008)have been previously shown. Importantly E16 immediately follows theperiod of neurogenesis of hippocampal pyramidal cells (E11-E15.5)(Rodier 1980). Thus we hypothesized that middle second trimesterinfection (E12.5, E15 and E17) in mice would alter brain cytokineexpression of the offspring since Fatemi and Folsom 2009 found thatinfection at E16 leads to altered expression of many brain genes in thehippocampi of the exposed mouse offspring.

Our results showed that the adult offspring of IL-6 treated animalsdisplayed behavioral deficits in social interaction and regulation ofanxiety that are reminiscent of autism. We also found neuropathologypreviously described (Turner 1999) including increase pro-inflammatorycytokine release and increased STAT3 phosphorylation. In many previousworks by other groups the pre-pulse inhibition (PPI) test isparticularly informative for animal models of autism, because there areexisting models for understanding the neural circuitry of startlegating; a process known to involve inhibitory cortico-striatal neuralcircuits (Braff et al. 2001). Furthermore, sensorimotor gating deficitshave been reported in a family of neurodevelopmental disorders and arenot specific to autism. Whereas Ornitz et al. 1993 reported equivocalPPI results in autistic children, others reported PPI deficits in adultswith Asperger syndrome (McAlonan et al. 2002), children with Tourettesyndrome (Swerdlow et al 2001), and men with fragile X syndrome(Frankland et al. 2004). To date, only one published study on adultswith autism (14 adult men diagnosed with autism and 16 typicallydeveloping normal comparison (NC) participants) has been conducted onthe subject and it concluded that PPI deficits may only be indirectlylinked to one of the hallmark features of autism. With this in mind wedid not measure PPI in this study because if it were normal, it wouldhave no scientific impact on the validity of the model, and it if wereabnormal, it in itself would not be a measure of true neuropsychiatricimpairment, rather the functional behavioral tests we performed would.

As a confirmation of our results, we examined the effects of luteolinand diosmin in regulating IL-6 induced JAK2/STAT3 phosphorylation invitro and if this could attenuate the pathologies and behavioraldeficits previously described. When murine N2a cells and primarycultured neuronal cells were co-treated with IL-6 and luteolin, weobserve a concentration dependent decrease in JAK2/STAT3phosphorylation, as evidenced by Western blot analysis of cell lysates.We next examined the in vivo effects of diosmin, with the STAT3inhibitor S31-201 as a positive control. When pregnant mice wereco-treated with IL-6 and either diosmin, or S31-201, we observed anattenuation of the behavioral deficits in the adult offspring of diosminand S31-201 co-treated animals as they showed social behaviorscomparable to that of control mice. Furthermore, when brain homogenatesof these adult offspring were examined, we saw decreased STAT3phosphorylation with decreased pro-inflammatory cytokine secretion.

Taken together, our results show that IL-6 induced JAK2/STAT3phosphorylation plays an integral role in the execution of IL-6/MIAmediated pathological effects. Indeed, inhibition of thisphosphorylation was able to attenuate both behavioral deficits andpathological outcomes such as increased inflammation.

Treatment with diosmin is not only effective, but safe. Indeed diosminis a natural flavonoid isolated from various plant sources or derivedfrom the flavonoid hesperidin. First used as a therapy in 1969, diosminis currently considered a vascular-protecting agent and has been usedfor treatment of chronic venous insufficiency/varicose veins (Jantet2002), hemorrhoids (Jantet 2000), lymphedema (Pecking et al. 1997), anddiabetes (Lacombe et al. 1989). The compound also exhibitsanti-inflammatory, antioxidant, and antimutagenic properties (Kuntz etal. 1999). Furthermore, marketed formulations 90% diosmin, 10%hesperiden pose little to no side effects. Taken together, there isconvincing evidence from preliminary studies regarding efficacy, as wellas published studies regarding safety in humans, that diosmin is a safeand potentially efficacious treatment for autism. Furthermore, the dosefound to be effective in this animal model (10 mg/kg/day) translatesinto a human dose of 0.81 mg/kg/day which is approximately 48.6 mg/dayfor a 60 kg person. While not reasonably achievable through consumptionof foods containing diosmin, this concentration may be provided througha daily oral supplement.

It will be seen that the advantages set forth above, and those madeapparent from the foregoing description, are efficiently attained andsince certain changes may be made in the above construction withoutdeparting from the scope of the invention, it is intended that allmatters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall there between. Now that theinvention has been described,

1. A method of treating an autoimmune disorder selected from the groupcomprising autism, schizophrenia, and diabetes comprising administeringa therapeutically effective amount of a flavonoid selected from thegroup comprising luteolin, diosmin, and diosmetin, their analogues,derivatives, and combinations thereof.
 2. The method of claim 1 whereinthe flavonoid is administered orally.
 3. The method of claim 1 whereinthe flavonoid is administered at a dosage of between about 0.81mg/kg/day and about 2.5 mg/kg/day.
 4. The method of claim 1 wherein theflavonoid is administered at a maximum dosage of about 2000 mg.
 5. Themethod of claim 1 wherein the flavonoid is administered at a dose of atleast 0.81 mg/kg/day.
 6. A method of treating autism comprisingadministering a therapeutically effective amount of a flavonoid selectedfrom the group comprising luteolin, diosmin, and diosmetin, theiranalogues, derivatives, and combinations thereof.
 7. The method of claim6 wherein the flavonoid is administered orally.
 8. The method of claim 6wherein the flavonoid is administered at a dosage of between about 0.81mg/kg/day and about 2.5 mg/kg/day
 9. The method of claim 6 wherein theflavonoid is administered at a maximum dosage of about 2000 mg.
 10. Themethod of claim 6 wherein the flavonoid is administered at a dose of atleast 0.81 mg/kg/day.
 11. A method of treating inflammation due to anautoimmune disorder selected from the group comprising autism,schizophrenia, and diabetes comprising administering a therapeuticallyeffective amount of a flavonoid selected from the group comprisingluteolin, diosmin, and diosmetin, their analogues, derivatives, andcombinations thereof.
 12. The method of claim 11 wherein the flavonoidis administered orally.
 13. The method of claim 11 wherein the flavonoidis administered at a dosage of between about 0.81 mg/kg/day and about2.5 mg/kg/day
 14. The method of claim 11 wherein the flavonoid isadministered at a maximum dosage of about 2000 mg.
 15. The method ofclaim 11 wherein the flavonoid is administered at a dose of at least0.81 mg/kg/day.